The migratory flights of birds are primarily fueled by fat; however, certain fatty acids may also enhance flight performance and the capacity to oxidize fat. The natural doping hypothesis posits that n-3 long-chain polyunsaturated fatty acids (PUFA) increase membrane fluidity and aerobic and fatty acid oxidative enzymes in the flight muscles, which enables prolonged endurance flight. Support for this hypothesis is mixed, and there is no empirical evidence for increased flight performance. We fed yellow-rumped warblers ( Setophaga coronata coronata) diets enriched in either n-3 or n-6 long-chain PUFA or low in long-chain PUFA and evaluated flight muscle metabolism and endurance performance in a wind tunnel flights lasting up to 6 h. Fatty acid profiles of muscle phospholipids confirmed enrichment of the targeted dietary fatty acids, whereas less substantial differences were observed in adipose triacylglycerol. Contrary to the predictions, feeding n-3 PUFA decreased peroxisome proliferator-activated receptors-β mRNA abundance and muscle oxidative enzyme activities. However, changes in muscle metabolism were not reflected in whole animal performance. No differences were observed in flight performance among diet treatments in terms of endurance capacity, energy costs, or fuel composition. These measures of flight performance were more strongly influenced by body mass and flight duration. Overall, we found no support for the natural doping hypothesis in a songbird. Furthermore, we caution against extending changes in flight muscle metabolic enzymes or fatty acid composition to changes to migratory performance without empirical evidence.